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Chapter 4 / 49

4 Steroid Hormones

Derek V. Henley, PhD, Jonathan Lindzey, PhD, and Kenneth S. Korach, PhD

CONTENTS INTRODUCTION STEROID SYNTHESIS MECHANISMS OF ACTION AND DEVELOPMENT STEROIDS AND NORMAL PHYSIOLOGY STEROIDS AND PATHOPHYSIOLOGY CONCLUSION

1. INTRODUCTION Steroids are lipophilic molecules used as chemical of steroid synthesis, steroid hormone effects in normal messengers by organisms ranging in complexity from physiology, molecular and biochemical mechanisms water mold to humans. In vertebrates, steroids act on a of action of steroid hormones, and pathologic states wide range of tissues and influence many aspects of related to steroid hormone action. biology including sexual differentiation, reproductive physiology, , and intermediate metabo- 2. STEROID HORMONE SYNTHESIS lism. Major sites of steroid synthesis and secretion Steroid hormones are molecules derived from include the , testes, adrenals, and . a common precursor (Cholestane, C27). Based on the distance of a target site from the site of There are four major classes of steroid hormones: synthesis and secretion, steroid hormones can be clas- progestins, , , and corticoids, sified as either endocrine (distant target tissue), which contain 21, 19, 18, and 21 carbons, respectively. paracrine (neighboring cells), or autocrine (same cell) Steroid hormones are synthesized by dehydrogenases factors. When secreted into the environment, steroids and P450 , which catalyze hydro- can also act as by conveying information xylation and dehydroxylation-oxidation reactions. to other organisms. Eukaryotic P450 are membrane-bound Owing to the pervasive effects of steroids in verte- enzymes expressed in either the inner mitochondrial or brate biology, a number of pathologic states can occur membranes of steroid-synthe- because of problems related to steroid hormone action sizing tissues. A common and important rate-limiting (see Section 6). These disease states include cancer, step for the synthesis of all steroid hormones is cleav- steroid insensitivity, and abnormal steroid synthesis. age of the side chain from cholesterol (C27) to yield The purpose of this chapter is to provide an overview (C21), the common branch point for synthesis of progestins, corticoids, androgens, and, From: : Basic and Clinical Principles, Second Edition hence, estrogens (Fig. 1). Expression of the side-chain (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ cleavage cytochrome P450scc (cytP450scc), 49 50 Part II / Hormone Secretion and Action

Fig. 1. (A) Synthetic pathways and structures of major progestins and corticoids found in humans. Major enzymes involved in the synthesis are in boldface.

which converts cholesterol to pregnenolone, is one of hormones from the such as follicle- the unique features of steroidogenic cells that partici- stimulating hormone (FSH), (LH), pate in de novo steroid synthesis. and adrenocorticotropic hormone (ACTH). Mineralo- In vertebrates, the synthesis and secretion of gonadal corticoids are also regulated by ion concentrations and and hormones are regulated by tropic circulating levels of II. Common regulatory Chapter 4 / Steroid Hormones 51

Fig. 1. (B) Synthetic pathways and structures of major androgens and estrogens found in humans. Major enzymes involved in the synthesis are in boldface.

mechanisms for steroid synthesis and release are nega- itary. The complex interplay among different compo- tive feedback loops in which elevated circulating levels nents of the hypothalamic-pituitary- (HPG)/adre- of steroids suppress production of tropic hormones by nal (HPA) axes is an important feature of endocrine acting at specific sites in the brain and the anterior pitu- physiology and is discussed in Section 5. 52 Part II / Hormone Secretion and Action

2.1. Synthesis of changes during the course of folliculogenesis Pregnenolone serves as a principal precursor to all during which, under the influence of LH, the thecal cells the other steroid hormones synthesized by the , synthesize and secrete and testoster- testes, or adrenals. It appears that the rate-limiting step one, which diffuse across the basement membrane and for the synthesis of progesterone is side-chain cleavage are subsequently aromatized to and E2, respec- of cholesterol by P450scc. Pregnenolone is then con- tively, by the granulosa cells. The level of verted into progesterone by 3β-hydroxysteroid dehy- and, hence, estrogens produced in the granulosa cells is drogenase (3β-HSD). Thus, deficiencies in either under the control of FSH during midfollicular phases. P450scc or 3β-HSD have profound effects on the syn- Later in the cycle, the follicle/corpora lutea express thesis of all steroids. greater numbers of LH receptors and LH begins to regu- In the ovary, progesterone is produced at all stages of late E2 production. During pregnancy, the placenta uti- follicular development as an intermediate for lizes androgen precursors from the fetal and estrogen synthesis but becomes a primary secretory and secretes large amounts of E2. In addition, in male product during the peri- and postovulatory (luteal) vertebrates, many target tissues such as pituitary cells phases. The synthesis of progesterone is under the con- and hypothalamic neurons convert circulating testoster- trol of FSH during the early stages of folliculogenesis one into E2. and, following acquisition of LH receptors, becomes 2.4. Synthesis of Corticoid sensitive to LH later in the ovarian cycle. The synthesis of progesterone by the is stimulated dur- Corticoids are divided into gluco- and mineralocor- ing early pregnancy by increasing levels of chorionic ticoid hormones. The predominant human glucocorti- . In addition, the placenta secretes high lev- coid, , is synthesized in the of els of progesterone during pregnancy, although a differ- the . The synthesis of cortisol involves α ent isozyme of 3β-HSD is involved in the synthesis. hydroxylations of progesterone at the 17 , 21 (CYP21), and 11β (CYP11B1) positions. The synthesis of cortisol 2.2. Synthesis of Androgen is under the control of an anterior pituitary hormone, Androgens are synthesized and secreted primarily by ACTH, and a negative feedback mechanism in which the Leydig cells of the testes, thecal cells of the ovary, elevated cortisol suppresses the release of ACTH (see and cells in the reticularis region of the adrenals. In most Section 5.2). tetrapod vertebrates, is the dominant circu- The dominant human is aldoster- lating androgen. Testicular synthesis and secretion of one, which is produced in the of the testosterone is stimulated by circulating LH, which adrenal. The synthesis of involves the syn- upregulates the amount of 17α-hydroxylase:C-17,20- thesis of and subsequent hydroxylation and lyase, a rate-limiting enzyme for conversion of C21 into oxidation at C18 to yield aldosterone. The synthesis of C19 steroids. Once taken up by target tissues, testoster- aldosterone is regulated directly by levels of , one can be reduced by 5α-reductase to yield a more and indirectly by the effects of levels and blood active androgen metabolite, 5α- volume on levels of angiotensin II (see Section 5.2). (5α-DHT). Testosterone and androstenedione can also β 2.5. Serum-Binding be converted into estrogens such as 17 - (E2) or estrone through a process termed aromatization. Following synthesis, steroids are transported to their Aromatization is carried out by a cytochromeP450 target tissues through the bloodstream. The hydropho- aromatase enzyme that is expressed in the granulosa bic nature of steroid hormones results in low water solu- cells of the ovary, Leydig cells of the testes, and many bility; therefore, transport proteins, known as other tissues including the placenta, brain, pituitary, serum-binding proteins, help transport steroid hormones , and . Indeed, many of the effects of to their target tissues. This transport is accomplished circulating testosterone are owing to conversion into through the binding of steroid hormones to a specific α either 5 -DHT or E2 within target tissues. high-affinity -binding domain (LBD) within the serum-binding proteins. Five serum-binding proteins 2.3. Synthesis of Estrogen have been identified: -binding globulin, Estrogens and progestins are synthesized and secreted retinol-binding , –binding globulin primarily by maturing follicles, corpora lutea of ova- (SHBG), thyroxine-binding globulin, and – ries, and the placenta during pregnancy. The predomi- binding protein. As indicated by their respective names, nant estrogen secreted is E2 and the predominant each serum-binding protein preferentially binds a progestin is progesterone. The profile of the synthesis of unique class of steroid hormones. Chapter 4 / Steroid Hormones 53

Table 1 Hormone Response Elements a Type of response element Sequence Species • Estrogen GGTCAcagTGACC vitA2 Xenopus GGTCAcggTGGCC PS2 Human GGTCAnnnTGACC Consensus • Androgen AGAACAgcaAGTGCT PSA Human • Progesterone AGTACGtgaTGTTCT C(3) Rat • AGA/GACAnnnTGTA/CCC/T Consensus • Mineralocorticoid

a Sequence of some characterized response elements for ERs vs ARs, PRs, and corticoid receptors are given. Also provided are consensus sequences for an ERE and a GRE (GRE consensus sequence is identical to a PRE and an ARE). Italicized nucleotides demonstrate potential sites for mutation that can convert one class of 4 to another.

Recent studies have suggested that serum-binding pro- in the nucleus, and glucocorticoid (GR) and teins may serve more dynamic roles beyond steroid hor- androgen receptor (AR) are located in the cytoplasm. mone transport. SHBG, e.g., has been shown to play a Current dogma holds that steroid hormones move pas- role in –associated sively from the circulation and interstitial spaces across through the second-messenger cyclic adenosine mono- cell membranes and bind to and activate NHR proteins. phosphate (cAMP) and protein kinase A (PKA). In addi- The activated NHR-ligand complex then associates with tion, cell-surface SHBG receptors have been identified in members of a class of signal modulators termed tissues such as the , testis, and , further coregulator proteins. The NHR-ligand-coregulator supporting a role for SHBG in . complex binds to specific DNA sequences termed hor- mone response elements (HREs) that are associated with 3. MECHANISMS OF STEROID promoter regions involved in regulating gene transcrip- HORMONE ACTION tion. Most ligand-bound NHR complexes bind to DNA The effects of steroids are typically slow in relation to as homodimers, although some NHRs, including vita- the rapid time courses for the effects of second-messen- min D and orphan receptors, can bind to DNA as heter- ger-mediated hormones. This is owing both to the odimers with other receptors such as the retinoid X signal amplification inherent to second-messenger cas- receptor. Binding of the activated NHR-ligand com- cades and to the slower changes in gene transcription and plexes to an HRE is thought to position the activated translation exerted by steroids (genomic effects). Early NHR so that transactivation domains of the NHR inter- experiments confirmed these paths of nuclear hormone act with proteins comprising the transcriptional com- action by utilizing protein and RNA synthesis inhibitors plex bound to a promoter and, hence, stimulate or inhibit such as cycloheximide and actinomycin D, respectively. rates of transcription. Though most characterized effects of nuclear hormones HREs are a family of highly related DNA palin- are mediated via nuclear receptors and genomic path- dromic repeats. The estrogen, COUP factor, ways, there are examples of very rapid, “nongenomic” hormone, and retinoic acid receptors share highly effects of steroids that appear to be owing to membrane- homologous consensus response elements, and GR, mediated effects. In addition, alternative mechanisms of AR, PR, and mineralocortoid receptor (MR) share very nuclear (NHR) activation include similar and, in some cases, identical elements. The high ligand-independent activation and genomic activation degree of homology between and within these two independent of a hormone-responsive element. groups of HREs is also reflected in the high degree of homology between protein sequences of the DNA- 3.1. Genomic Mechanisms binding domains (DBD) of the various receptors. This of Steroid Action would seem to create a problem with specificity of The basic genomic mechanisms of steroid action hold hormone action but, as seen in Table 1, mutation of two relatively constant across different target tissues and nucleotides is sufficient to alter a consensus estrogen different classes of nuclear hormones despite the wide response element (ERE) into a consensus androgen diversity in target tissues and the responses elicited. In response element. In addition, as other nonconsensus the absence of hormone, (ER) and elements are characterized more light is shed on the progesterone receptor (PR) are principally localized nature of NHR-specific interactions with the . 54 Part II / Hormone Secretion and Action

Fig. 2. Mechanisms of nuclear hormone action. E2 and ER-mediated biologic effects occur through multiple pathways. 1. In the classic ligand-dependent pathway, E2 diffuses across the cell membrane and binds to ER, causing dissociation of heat-shock proteins and allowing the activated ligand-ER complex to recruit transcriptional coactivators and bind to an ERE, resulting in the up- or downregulation of gene transcription. 2. Ligand-independent ER activation occurs following (GF) stimulation of kinase pathways that phosphorylate the ER. 3. E2-ER complexes can transactivate in an ERE-independent manner through association with other DNA-bound transcription factors. 4. E2 can exert rapid effects on a cell through nongenomic actions that occur at the cell surface.

The different classes of steroid hormones are all 3.2. Structure of ER Gene and Protein present in the circulation, and their respective levels Two forms of the ER have been identified, ERα and vary with the different physiologic states of the organ- ERβ, that are coded for by separate genes located on ism. In addition, many target cells express multiple separate chromosomes. Both ER proteins contain modu- classes of NHR. This presents the organism with the lar functional domain structures characteristic of the problem of how to activate a specific gene by a specific steroid hormone superfamily. The ER steroid hormone. Specificity of steroid hormone– acti- proteins contain six functional domains that are termed vated gene expression lies in (1) hormone-specific bind- A/B, C, D, E, and F domains. These domains have been ing by the receptor, (2) DNA-specific binding exhibited found to possess the following functions: ligand-inde- by the different types of steroid receptors, and (3) con- pendent activation function (A/B), DNA binding (C), trol of access of steroid receptors to genes through dif- ligand binding (E), nuclear localization (D), and dimer- ferential organization of chromatin in the many different ization and ligand-dependent activation function (E) target cells and tissues. Many of the hormone insensitiv- (Fig. 3). The ERα and ERβ proteins share a high degree ity syndromes stem from mutations that alter steroid- or of homology within their DBDs and LBDs, 97 and 60%, DNA-binding characteristics of the NHR. respectively, which results in both receptors binding to As a whole, NHR proteins are a highly conserved the same EREs and exhibiting a similar binding affinity group of “ligand-dependent” nuclear transcription fac- for most endogenous and exogenous ER ligands. The tors (Fig. 2). NHRs are modular in nature and can be modular nature of the different functional domains and broken down into different functional domains such as the interdependency of these domains means that splice transactivating domains, DBD, and LBD. Among the variants of NHR mRNAs can produce altered proteins different classes of NHRs—AR, PR, ER, GR, and MR, that behave in appreciably different fashions from the the DBD is the most highly conserved region followed full-length NHR. The importance of these variants in by the LBD and then the amino-terminal transactivating normal physiology is still under investigation, but splice domain. The following discussion of different func- variants may play a role in disease states such as the tional domains focuses on the ER, but many of the char- progression from steroid-dependent to -independent acteristics hold true for other NHR types. cancer (see Section 6.1). Chapter 4 / Steroid Hormones 55

molecule contacting each 5-bp inverted repeat. Binding of the ER-ligand complex to an ERE sequence positions the ligand-activated ER and associated coactivators where they can interact with the basal transcription com- plexes and influence the rate of gene transcription. In addition to ERE-mediated gene expression, recent evi- dence indicates that the ERs are capable of transacti- vating genes whose promoters lack a functional ERE through protein-protein interactions with other DNA- bound transcription factors, such as Fos and Jun, at AP-1-binding sites. The result of the ER association is Fig. 3. Protein structure of ERs. Major functional domains of the a tethering of the ER to DNA and an upregulation of α β mouse ERs and the homology between ER and ER with re- gene expression via an ERE-independent mechanism. spect to these domains is shown. 3.2.3. TRANSCRIPTION ACTIVATION FUNCTIONS ERα contains two regions known to possess tran- scriptional activation functions, activation function-1 3.2.1. LIGAND-BINDING DOMAIN (AF-1) and AF-2, located in the A/B and E domains, The LBDs (domain E) of ERα and ERβ consist of respectively. Depending on the cell type and target 251 and 245 amino acids, respectively, and are coded genes, AF-1 and AF-2 can act independently or in con- for by exons 5–9. The LBD forms a large hydrophobic cert. For instance, removal of AF-1 has no effect on E2 pocket that exhibits specific, high-affinity binding for induction of a reporter construct containing the vitellogenin ERE, whereas the same AF-1 deficient ERα estradiol (kd ~ 0.1 nM). Binding of estrogens to this re- gion produces a conformational change in the ER that has only 20% of the wild-type induction of a PS2-ERE. allows for the recruitment of transcriptional coregulators As mentioned earlier, removal of the LBD (containing α and subsequent transcriptional activation or suppres- AF-2) can lead to a constitutively active ER . Interest- sion of target genes. Based on studies in which removal ingly, this constitutive activity may require phosphory- of the LBD results in a constitutively active or “ligand- lation and activation by second messengers. Studies α independent” ER, it is possible that the LBD functions using AF-1 and AF-2 truncated ER have demonstrated as a repressor of a transcription factor that would nor- that AF-1 responds to growth factors that act via second mally be constitutively active. Indeed, a constitutively messengers such as cAMP or to mitogen-activated pro- active exon 5 splice variant of ERα has been detected in tein kinase (MAPK) signaling pathway activation, whereas AF-2 is E (ligand) dependent. Thus, the ER is some human breast cancers. Finally, it appears that E2 2 binding to the LBD of the ER is not always necessary for actually a nuclear transcription factor that responds to ER-mediated genomic actions. Recent evidence has both steroid and second-messenger signaling pathways. shown ligand-independent ER activation of target genes In contrast to the well-characterized activation domains α β owing to growth factor activation of kinase signaling of ER , the roles of the homologous regions of ER pathways. have not been clearly defined with respect to transcrip- tional activity. “Ligand-independent” or second-mes- 3.2.2. DNA-BINDING DOMAIN senger activation of transcriptional activity has also been The DBD exhibits specific binding for sequences of demonstrated for AR and PR, suggesting that this may DNA termed EREs. This region is highly conserved and be an important and conserved mechanism for physi- contains two “zinc finger” motifs, each of which con- ologic activation of steroid receptors. tains cysteine residues that bind zinc. The first zinc fin- The transcriptional activation functions of AF-1 and ger dictates sequence-specific interactions with DNA, AF-2 are mediated through transcriptional coregulators, and the second appears to dictate the spacing require- proteins that provide the link between ligand-activated, ments between the arms of the palindrome. These fin- DNA-bound receptors and the general transcriptional gers are critical for DNA binding but the surrounding machinery. The conformational change induced by amino acids also influence binding. The canonical agonist binding to the ER allows coregulators to inter- element is a palindrome inverted repeat (GGTCA act primarily with AF-2 sites on the receptor; however, nnnTGACC) although deviations from this consensus interaction with AF-1 sites does occur. Many different sequence are quite common (see Table 1). The ER binds coregulators have been identified that interact with the to the DNA sequence as a dimer with one receptor ligand-bound ER, including the p160 family members 56 Part II / Hormone Secretion and Action

SRC-1, GRIP1, and AIB1, p68 helicase, and CBP/p300. oocytes, all of which exhibit increased intracellular cal- The p160 family of coregulators contains characteristic cium concentrations shortly, if not immediately, after α -helical LXXLL motifs that are involved in AF recog- E2 exposure. Other estrogen-mediated nongenomic nition and binding. mechanisms have been observed in spermatozoa, breast cells, nerve cells, and vascular tissues. In addition, 3.2.4. DIMERIZATION nongenomic mechanisms have been described for Most data indicate that NHRs act as homodimers, progesterone, androgens, , and miner- although some data suggest possible effects by NHR alocorticoids. Current research is under way to deter- monomers. The region of the protein responsible for mine whether these nongenomic steroid mechanisms dimerization of the mouse ER overlaps with steroid- are owing to receptor-independent events at the plasma binding function and spans amino acids 501–522. These membrane, nonsteroid associated membrane receptors, amino acids form an amphipathic, helical structure with or membrane-bound NHRs. an imperfect heptad repeat of hydrophobic amino acids reminiscent of the leucine zippers found in the JUN/ 4. STEROIDS AND DEVELOPMENT FOS and CREB families of transcription factors. Muta- Scientists have known for years that in utero and tions of amino acids in this hydrophobic stretch have neonatal exposure to steroids are critical for sexual dif- proven that this area is critical for dimerization, steroid ferentiation of the brain and peripheral reproductive binding, and, hence, transactivation. The dimerization structures. A guiding concept for the study of develop- function is critical for the effects of NHR homodimers mental actions of steroidal effects is the organization- but may also play a role in the formation of hetero- activation hypothesis. Stated simply, prenatal or dimers between NHRs and other transcription factors. neonatal exposure to steroid hormones organizes or Heterodimers consisting of ERα and ERβ, as well as alters differentiation of the phenotype such that hor- heterodimers of ERα and SP1 proteins, have been shown monal exposure in adulthood is more likely to activate to regulate expression of genes such as c-FOS and trans- a particular response. A corollary of this rule is that the forming growth factor-α. Thus, the dimerization func- initial exposures must fall within certain critical peri- tion is critical for the effects of NHR homodimers ods of sensitivity. These critical periods typically occur but also plays a role in the formation of heterodimers during the fetal, neonatal, and pubertal stages. between NHRs and other transcription factors with simi- Steroids affect development of organs and tissues lar dimerization domains. through both induction and inhibition of growth. Inhibi- tion occurs via active cell death, a process termed 3.2.5. NUCLEAR LOCALIZATION SIGNAL . Apoptosis is an active process requiring pro- NHRs and many other transcription factors possess a tein synthesis and resulting in chromatin condensation, segment of amino acids that targets the proteins to the degradation of chromatin in a characteristic segmented . These stretches of amino acids tend to be manner that produces an observable “ladder” pattern, basic and have been termed the nuclear localization and development of apoptotic bodies. signal (NLS). It appears that the NLS is located between amino acids 250 and 270 of the ERα, a region that shares 4.1. Stromal-Mesenchymal Interactions homology with the nuclear localization domains of the A recurring theme in development of steroid-depen- glucocorticoid and progesterone receptors. The NLS for dent glandular tissues is the importance of stromal- ERβ has yet to be characterized. mesenchymal tissue induction. In this scheme, the fate of undifferentiated epithelium is determined by the 3.3. Nongenomic Mechanisms underlying mesenchyme with which it comes into con- of Steroid Action tact. For instance, undifferentiated epithelium com- Although steroids typically act through the classic bined with prostatic or integumental mesenchyme genomic mechanism, a process that takes several min- develops a phenotype dictated by the type of mesen- utes to hours for effects to be seen, steroids are also chyme. In the case of hormone-directed morphogen- capable of eliciting rapid biologic effects within sec- esis such as in the prostate or breast, hormonal onds to minutes after administration through nonge- influences on the glandular epithelium can occur either nomic mechanisms. Nongenomic steroid action results directly on epithelial cells or indirectly via inductive in the rapid activation of a variety of cell-signaling influences of the mesenchyme. Recent experiments molecules, including MAPKs, adenylyl cyclase, and demonstrate that epithelium can also influence the PKA and PKC. Rapid responses to estrogen have been underlying mesenchyme, indicating a bidirectional observed in granulosa cells, endometrial cells, and epithelial-mesenchymal interaction. Chapter 4 / Steroid Hormones 57

4.2. Secondary Sex Structures medial preoptic area of the and in the In the developing mammalian embryo, gonadal sex spinal cord. The development of these nuclei and subse- is determined by genotype. In turn, the embryonic quent function in adult males are androgen dependent; secrete hormones that, coupled with maternal androgen ablation during early critical periods of differ- hormones, determine the early hormonal milieu to entiation leads to smaller, female-typical nuclei and also which secondary sex structures are exposed and, hence, decreases in male-typical copulatory behavior. In rats, dictate development of male or female phenotype. the effects of testicular testoterone on the sexually di- Dogma holds that mammals possess a default system morphic nuclei of the medial preoptic area appear to be such that embryos develop a female phenotype in the predominantly through aromatization to E2; treatment absence of any gonadal steroid hormones. In males, as with E2 mimics the effect of testosterone, and the use of the developing testes begin to develop sex cords, the an aromatase inhibitor can prevent masculinization of testes secrete Müllerian-inhibiting substance (MIS) and sexually dimorphic nuclei. Similar steroid-dependent testosterone. The MIS induces ipsilateral regression of dimorphisms are found in the CNSs of gerbils, voles, the Müllerian ducts, which prevents development of songbirds, lizards, and fish. These dimorphisms may be Müllerian derivatives such as the uterus and fallopian present as differences in gross volume, cell number; cell tubes. Elevated testosterone stimulates development of size, dendritic arborization, and levels of expression of Wolffian derivatives such as epididymis, vas deferens, enzymes, neurotransmitters, neuropeptides, or recep- and seminal vesicles. Differentiation of external geni- tors. Sexual dimorphisms in humans have also been talia and accessory glands (such as the prostate) from reported in the anterior hypothalamus (AH), preoptic the genital tubercle, scrotal folds, and urogenital sinus area (POA), and anterior commissure, although there requires 5α-DHT. This is illustrated by 5α-reductase- are some conflicting data. deficient males who have normal Wolffian derivatives but have feminized external genitalia despite the pres- 4.4. Steroids and Bone ence of testosterone (see Section 6.2). Bone cells express ER, AR, and PR and the develop- Although it is true that external genitalia and internal ment and maintenance of bone structure is regulated by reproductive structures of genotypic females are grossly estrogens and androgens. Pubertal surges in estrogens feminized without the influence of gonadal steroids, it and androgens initiate growth spurts including long bone is clear that steroidal effects are needed for complete growth, primarily mediated by increased -like and functional differentiation of some structures such as growth factor-1, and, subsequently, cessation of bone the uterus and breast. For instance, gene-targeted mice growth through epiphyseal closure. In adults, E2 main- lacking functional ERα (αERKO) have uteri that pos- tains bone mass and mineralization. The importance of sess all the normal tissue types and structures but are the effects of E2 on bone growth and development is hypoplastic. In addition, in wild-type females, exposure manifest in individuals lacking in E2 action. For instance, to estrogens and progestins is required for differentia- a human male patient lacking functional ERα exhibits tion of the nipple and mesenchyme surrounding the continued bone growth, decreased bone density, and epithelium of breast tissue. Estrogen and progesterone absence of epiphyseal closure (see Section 6.3). In addi- also increase alveolar formation and branching of mam- tion, the absence of E2 owing to either ovariectomy or mary ducts during mammary gland development. menopause contributes to osteoporosis whereas exogen- ous E2 helps ameliorate this condition. Excess produc- 4.3. Sexual Behavior and Sexual tion of cortisol results in a loss of bone mass (osteopenia). Dimorphisms of Brain Sex behavior in most adult vertebrates is dependent 4.5. Steroids and Liver on (1) organizational effects of hormones early in devel- Liver cells express ERs and ARs which regulate pro- opment, and (2) activational effects of circulating ste- duction of secreted proteins and steroid-metabolizing roids in the adult. In many species, in utero and neonatal enzymes. In humans, the liver synthesizes and secretes hormone exposures alter adult patterns of sexual behav- into the bloodstream a plasma protein termed SHBG. iors. Historically, this observation led to the assumption This protein serves to sequester and prevent steroids that at some organizational level the brains of males and from being metabolized and/or cleared from the blood- females must be morphologically or functionally dis- stream. SHBG binds DHT with high affinity (kd ~ 0.5 tinct in order to favor female- or male-typical behaviors. nM) and testosterone and E2 with approx 5- and 15-fold In the case of the rat, sexually dimorphic nuclei have lower affinity, respectively. Estrogens stimulate been found in the central nervous system (CNS). Male whereas androgens inhibit the synthesis and secretion of rats possess enlarged sexually dimorphic nuclei in the hepatic SHBG. 58 Part II / Hormone Secretion and Action

into the hypophysial portal system, which carries GnRH to the pituitary. GnRH stimulates synthesis and release of LH and FSH, which, in turn, regulate steroidogenesis and gametogenesis. Elevations in the circulating con- centrations of gonadal steroids feed back on hypotha- lamic and pituitary sites to regulate gonadotropin synthesis and release. In addition to steroids, the gonads secrete peptide hormones () that feed back on the pituitary to regulate synthesis and release of gonadotropin. GnRH secretion occurs as both an episodic, tonic pattern and a surge associated with ovulatory events in females. The tonic pattern of GnRH secretion occurs in a pulsatile fashion with a periodicity of approx 1 pulse/ h. Steroids feed back on the GnRH pulse generator thought to be located in the medial basal hypothalamus to regulate tonic patterns of GnRH secretion. Regula- tion of the ovulatory GnRH surge, however, appears to require input from the POA/AH regions. In addition, androgens and estrogens can feed back directly on pitu- itary gonadotropes to regulate cell growth, sensitivity to Fig. 4. HPG/HPA axes. Depicted are the pathways and major GnRH, and basal levels of gene expression of gonado- tropic hormones involved in regulating the production of gonadal tropins. A final level at which steroids feed back is on and adrenal steroids. steroidogenic cells themselves. For instance, experi- ments indicate that androgens can downregulate LH- induced expression of steroids by Leydig cells. The There are distinct sex differences in the profile of feedback effects of steroids on these different levels steroid metabolites excreted in . The basis of such constitute long loop (gonad-hypothalamic), short loop sex differences results from sex differences in expres- (gonad-pituitary), and ultrashort loop (gonad-gonad) sion of metabolic enzymes in the liver. For instance, feedback circuits (Fig. 4). the female liver expresses 15α-hydroxylase activity whereas the male does not express this enzyme. By 5.1.1. FEMALE REPRODUCTIVE CYCLES contrast, males express 16α-hydroxylase, which is Humans and most other female mammals are sponta- absent in females. In rats, these sex differences are neous ovulators, that is, the cyclical buildup of estrogen regulated by what constitutes a hypothalamic-pitu- triggers a “spontaneous” pulse of gonadotropin that trig- itary-hepatic axis in which neonatal androgens gers ovulation independent of mating stimuli. However, masculinize the (GH) axis. In turn, in some species, females are reflex ovulators; that is the pattern of (GH) secretion imprints a male or female physical mating stimuli are responsible for triggering profile of steroid . This is evident by the GnRH and gonadotropin surges that provide the proxi- fact that pulsatile surges of GH (malelike pattern) or mate cues for ovulation. The following discusses nor- tonic, low-level GH infusions (femalelike) into hypo- mal ovarian cycles in spontaneous ovulators. physectomized rats produce a male- or female-typical As a follicle matures under the influence of basal pattern of enzyme expression and metabolism, respec- levels of FSH, circulating levels of E increase to a peak tively. 2 at or near ovulation. The increase in E2 increases 5. STEROIDS AND NORMAL PHYSIOLOGY gonadotrope sensitivity to GnRH by upregulating GnRH receptors and, at peak levels, exerts a positive feedback 5.1.HPG Axes effect that triggers a GnRH surge that, in turn, produces Gonadal function is regulated by the pituitary gona- an LH surge. This LH surge induces ovulation, forma- dotropins (LH and FSH), which are regulated by the tion of a corpora lutea from granulosa cells of the fol- hypothalamic peptide, gonadotropin-releasing hormone licle, and synthesis and secretion of progesterone and (GnRH), steroids, and gonadal (Fig. 4). GnRH E2. Over the course of the ovarian cycle, ovarian is synthesized by small populations of neurons in the steroids exert control over GnRH and , POA, AH, and mediobasal hypothalamus and is released maturation of follicles, preparation of the uterus for Chapter 4 / Steroid Hormones 59 implantation, alterations in vaginal and cervical func- rition and expulsion of the placenta causes an abrupt tion, and behavior. drop in E2 and progesterone. 5.1.1.1. Feedback. Long-term ovariectomy of mice 5.1.1.3. . Critical stages of sex determination leads to a large increase in steady-state message for and sexual differentiation occur in utero and early in FSH and LH, whereas estrogen treatments reverse this neonatal life. However, terminal differentiation of sexu- effect through a negative feedback mechanism. How- ally dimorphic structures and the onset of reproductive ever, this is an oversimplification of the complex fertility occur during puberty. In humans, the onset of effects of ovarian steroids on feedback regulation of the puberty is marked by an increase in tonic, pulsatile hypothalamus and pituitary. Estrogen appears to have GnRH release and increased secretion of LH and FSH. biphasic effects on the synthesis and secretion of LH In the female, increased gonadotropin levels initiate and FSH in which lower levels of E2 present during waves of folliculogenesis and associated increases in E2 postovulatory and early follicular phases can suppress and androgens. As levels of E2 increase, terminal differ- gonadotropins, whereas the higher levels of E2 found entiation of the begins and females undergo a during late folliculogenesis result in (1) increased sen- growth spurt. As E2 levels increase over the course of sitivity of gonadotropes to GnRH, and/or (2) a preovu- puberty, E2 induces epiphyseal closure and cessation of latory pulse of GnRH. Similarly, following E2 priming, the growth spurt. Exposure to increasing levels of E2 the initial exposure to progesterone results in increased results in an initial proliferation of the endometrium sensitivity to GnRH followed by long-term inhibition. followed by the first menses (menarche) owing to a drop Indeed, elevated progesterone associated with forma- in E2 at the end of a follicular wave. The initial ovulatory tion of the corpora lutea results in suppression of estrous event takes place approx 1 yr following menarche, pre- cycles. sumably because the mechanisms regulating a GnRH 5.1.1.2.Effects of Estradiol and Progesterone on surge now respond to E2-positive feedback. In the male, Accessory Sex Structures. As E2 levels increase during the pubertal onset of increased GnRH and gonadotropin the follicular phase, the luminal epithelium of the uterus synthesis and release is marked by testicular enlarge- enters a proliferative phase in preparation for implanta- ment and initiation of spermatogenesis and steroido- tion. In mice, initial E2 exposure rapidly induces (1–4 h) genesis. As levels of circulating testosterone increase, hyperemia and water imbibition followed by increases penile enlargement, growth of , and growth in DNA and protein synthesis, hyperplasia, and hyper- spurts commence. In addition, the glandular epithelium trophy. An important effect of E2 during this stage is the of secretory glands such as the seminal vesicle and pros- induction of synthesis of progesterone receptors, which tate undergo a proliferative phase and begin to produce allows the uterus to respond to elevated progesterone. secretory products that become components of the During luteal phases, the uterus enters a proliferative semen. phase during which elevated progesterone completes 5.1.1.4. Effects of E2 and Progesterone on Sexual the preparation of the endometrium for implantation of Behavior. In many vertebrates, E2 and progesterone act the blastocyst by increasing vascularization and by to coordinate periods of maximum sexual receptivity thickening the mucosal layer of the epithelium. In the with periods of maximum likelihood of fertilization. absence of implantation, the corpora lutea degenerate, Thus, E2 priming during follicular phases followed by a serum progesterone levels drop, and the endometrium surge of progesterone associated with ovulation and degenerates. The hypothalamic-pituitary-gonadal axis luteinization results in maximum receptivity near the is then freed from progesterone suppression to resume time of ovulation. The effects of the estrous cycle on another round of folliculogenesis. behavior can be re-created in ovariectomized female Mammary gland function is regulated by the coordi- rodents treated with E2 followed by progesterone. The nated actions of estradiol, progesterone, and E2 treatment has a facilitatory effect alone but is greatly (PRL). Estradiol promotes lobuloalveolar development augmented by subsequent progesterone treatment. by acting directly on the mammary gland and by stimu- Based on lesion studies and intrahypothalamic implants lating the synthesis and secretion of PRL by the anterior of E2 and progesterone, the ventromedial hypothalamus pituitary. Estradiol-stimulated increases in PRL also appears to be the site of E2 and progesterone effects on help prepare the glandular tissue for lactation. Proges- receptive and proceptive behaviors in female mammals. terone promotes glandular development but requires (1) One effect of E2 is to upregulate PR in the ventromedial the presence of pituitary hormones and (2) priming with hypothalamus. The significance of E2 and progesterone E2, which upregulates levels of PR. While progesterone in sexual behaviors of female humans appears less pro- and E2 help prepare the glandular tissue for lactation, found than in other mammals with a distinct behavioral these two hormones also suppress lactation until partu- estrus. 60 Part II / Hormone Secretion and Action

5.1.2. MALE REPRODUCTIVE CYCLES releasing factor (CRF), produced by the paraventricu- Regulation of the male HPG axis is a less dynamic lar nucleus of the hypothalamus. Increased levels of process in which GnRH pulses are lower in magnitude cortisol act directly at the level of the corticotroph to and do not undergo surges like those associated with reduce ACTH production and at the paraventricular ovulation in females. In rodents, the male GnRH sys- nucleus of the hypothalamus to suppress CRF levels (Fig. 4). Thus, hypophysectomy leads to a decrease in tem cannot respond to exogenous E2 with a surge, whereas in humans and monkeys injections of exogen- cortisol and adrenalectomy leads to an increase in ACTH that is reversed by cortisol treatment. Continu- ous E2 result in a GnRH surge. Thus, the absence of a GnRH surge in male humans is owing to the absence of ous ACTH the estrogen buildup associated with folliculogenesis secretion characteristic of chronic also leads to in females. adrenal cortical hypertrophy; elevated glucocorticoid GnRH stimulates gonadotropes to synthesize and levels; and, in some cases, adrenal failure. release FSH and LH, which act on spermatogenesis and ACTH has a very limited tropic effect on the zona steroidogenesis, respectively. LH elevates cAMP lev- glomerulosa and aldosterone levels in mammals. Pri- els, which stimulates synthesis and secretion of test- mary regulators of aldosterone synthesis include potas- + + osterone from the Leydig cells. Elevated testosterone sium (K ), sodium (Na ), , and angiotensin + assists spermatogenesis and feeds back to downregulate II. Elevated angiotensin II, K loading, and low serum + GnRH levels and, hence, synthesis and release of gona- Na levels, however, increase synthesis of adrenal al- + dotropins. It appears that testosterone may feed back dosterone, which promotes Na by the kid- + both directly as an androgen and as an estrogen follow- ney. Conversely, Na loading produces or ing aromatization. Indeed, recent studies with αERKO circulatory expansion and decreases levels of aldoster- males demonstrated that ERα as well as AR pathway one. can effectively suppress serum LH at levels of both the Potassium appears to act directly at the level of the + pituitary and hypothalamus. Gonadotropes express both glomerulosa both in vivo and in vitro. Furthermore, K AR and ERα whereas GnRH neurons do not express loading appears to affect the release of and large amounts of either AR or ERα. Thus, the hypotha- synergizes with angiotensin II to increase the release of lamic feedback probably occurs in an indirect fashion aldosterone. Sodium depletion decreases blood volume, through AR- and ERα-expressing neurons that inner- which stimulates the juxtaglomerular apparatus to se- vate the activity of GnRH neurons from a distant site. crete renin. In turn, renin cleaves angiotensinogen, pro- Sexual behavior in most adult male vertebrates is ducing angiotensin I and initiating a cascade that dependent on elevated circulating levels of testoster- eventually leads to elevated levels of angiotensin II and one. Testosterone acts on brain nuclei of the AH/POA subsequent elevation of aldosterone. The elevated al- + to activate male-typical sexual behaviors; lesions of dosterone promotes Na retention and elevates blood these brain areas or castration results in a cessation of volume and arterial pressure, which, in turn, feeds back sexual behaviors. Depending on the species, behavioral to decrease renin production. effects of androgens can be owing to both aromatiza- 5.2.1. CARBOHYDRATE METABOLISM tion to E2 and direct effects as testosterone or DHT. Testosterone also acts on Sertoli cells, where it can Adrenalectomy leads to reduced liver glycogen and maintain spermatogenesis, even in hypophysectomized low blood glucose resulting from increased oxidation of males. Another important function is stimulation of glucose and decreased gluconeogenesis from protein. accessory sex structures such as the prostate and semi- Conversely, administration of cortisol leads to a rise in nal vesicle. Circulating testosterone is converted into blood sugar and an increase in liver glycogen stores 5α-DHT, which causes hypertrophy of secretory epi- owing to decreased glucose utilization and increased thelium, increases in RNA and protein synthesis, and gluconeogenesis. also elevates glucose levels increased protein secretions. As adults, the continued and promotes glycogen breakdown. Insulin, however, functioning of these androgen-dependent responses has the opposite effects, producing lower blood sugar relies on exposure to circulating testosterone. (decreased gluconeogenesis) and increased glycogen synthesis and storage. 5.2. HPA Axis Synthesis and secretion of glucocorticoids such as 5.2.2. STRESS RESPONSES cortisol are stimulated by the pituitary hormone, Stress can be induced by social interactions, physi- ACTH. The release of ACTH from corticotropes is cal stress, and physiologic challenges. A classic stress stimulated by a hypothalamic peptide, corticotropin- response is increased secretion of glucocorticoids trig- Chapter 4 / Steroid Hormones 61

Table 2 Steroid-Based Pathologies Defect Phenotype AR X: feminization of external genitalia, androgen resistant 5α-Reductase XY: feminization of external genitalia ERα XX: mammary agenesis, normal Müllerian structures, elevated androgens and gonadotropins, polycystic ovaries, infertile Human XY: tall stature, open epiphyseal plate, elevated estrogens and gonadotropins ERβ XX: subfertile, normal mammary gland development and lactation XY: fertile, no testicular phenotype Aromatase XX: mammary agenesis, ambiguous external genitalia, normal Müllerian structures, elevated androgens and gonadotropins, polycystic ovaries XY: phenotypes similar to those from ER defects but responds to estrogen therapy PR A XX: impaired ovulation, impaired uterine decidualization, infertility PR B XX: reduced mammary ductal morphogenesis and alveologenesis P450scc XX: asteroidogenesis, hyponatremia, altered glucose metabolism, no pubertal changes XY: same as XX, external genitalia feminized, Wolffian structures absent 3β-HSD XX: similar to P450scc deficiency except some masculinization may be present XY: similar to P450scc deficiency 21-Hydroxylase Inability to synthesize cortisol XX: increased androgens, ambiguous genitalia, masculinization, rapid postnatal somatic growth XY: increased androgens, rapid postnatal somatic growth

gered by increased secretions of CRH and ACTH. B-cells. High levels of glucocorticoids can also lead to Hormonal responses to stress can be very fast (min- apoptosis of immune cells in the adult and, conse- utes) and dissipate quickly, or in some cases, become quently, a compromised immune system and increased chronic. In stressful situations, elevated glucocorti- susceptibility to disease. coids stimulate an adaptive rise in glucose levels 5.2.3. BALANCE from carbohydrate energy sources. The effects of long- Salt balance is achieved primarily by mineralocorti- term elevated glucocorticoid include suppression of coids and neurohypophyseal peptides such as arginine HPG function and suppression of the immune and arginine vasotocin. Aldosterone, the system. principal human mineralocorticoid, reduces Na+ loss by Elevated glucocorticoids suppress production of enhancing resorption by the renal tubules of the . GnRH by hypothalamic neurons and, consequently, Thus, adrenalectomy or deficiencies in adrenal steroid alters gonadotropin and steroid synthesis and synthesis result in rapid decreases in blood Na+ and cir- gametogenesis. Glucocorticoids also directly affect culatory collapse unless Na+ or exogenous aldosterone gonadotrope function by suppressing basal and sec- is provided. ond-messenger-induced synthesis and release of gona- dotropins. At the level of the gonad, glucocorticoids 6. STEROIDS AND PATHOPHYSIOLOGY also suppress gonadotropin and second-messenger- Steroid-related pathologies (Table 2) include non- stimulated steroid synthesis. Thus, the suppressive heritable steroid-dependent cancers and heritable syn- effects of elevated glucocorticoids on are dromes that affect the synthesis or function of steroids exerted at all levels of the HPG axis. and their receptors, resulting in steroid insensitivity Glucocorticoids play a role in the apoptotic events syndromes. Even though the effects of steroids on ste- leading to differentiation of the immune system and roid-dependent cancers are environmental and non- regulation of the immune system in adults. Apoptosis is heritable, there are clearly genetic predispositions to an active process of programmed cell death in which a developing such cancers. The heritable defects in ste- series of programmed events leads to the death of a roid action are generally autosomal recessive diseases cell. In the developing immune system, glucocorticoids that lead to developmental anomalies with various induce apoptosis in autoreactive T-cells and unreacted degrees of severity. 62 Part II / Hormone Secretion and Action

6.1. Cancer talia. In cases of enzymatic deficiencies, hormone A number of steroid-dependent and steroid-indepen- therapy can ameliorate some symptoms, whereas those dent tumors occur in steroid target tissues such as the symptoms related to receptor defects are resistant to uterus, breast, and prostate. In the case of prostate can- hormone therapy. cer, a clear link with androgens is provided by the fact 6.2.1. 5α-REDUCTASE DEFICIENCY that castrated males never develop . Fur- α thermore, many prostate cancers exhibit a period of 5 -Reductase type 1 is expressed at low levels in α regression and remission following castration and peripheral tissues, and 5 -reductase type 2 is expressed α treatment. Unfortunately, many of these at high levels in male genital structures. In males, 5 - cancers enter a steroid-independent stage during which reductase deficiencies result in varying degrees of am- growth and metastases are independent of androgens or biguity of the external genitalia ranging from hormonal therapy. hypospadias to complete feminization. Under the influ- A vital question is, Why does prostate cancer become ence of elevated testosterone, Wolffian derivatives such steroid independent? Since normal proliferation and as the epididymis and seminal vesicle develop normally growth cycles are dependent on androgens, the question whereas the external genitalia are feminized to varying becomes, Why do these tumor cells lose their normal degrees. In addition, Müllerian derivatives are absent requirement for androgen stimulation? Two hypotheses owing to production of MIS by the testes. In extreme seem viable: (1) splice variants result in a constitutively cases of feminization, this syndrome is often diagnosed active variant AR that stimulates growth independent of at the age of puberty when a patient with female pheno- androgens, and (2) key regulatory points in the cell cycle type exhibits amenorrhea and/or some increased mas- lose the requirement for androgen stimulation. These culinization owing to the increased levels of testosterone hypotheses remain to be tested in this and other steroid- associated with puberty. Prior to puberty, these indi- independent tumors. viduals are usually raised with female roles, but is often amenable to treatment with following pubertal changes in phenotype they some- tissue-specific steroid antagonists, including times assume male gender roles. and faslodex, as assessed clinically by assays for both 6.2.2. ANDROGEN INSENSITIVITY SYNDROME ER and PR in mammary biopsies. The presence of AND TESTICULAR FEMINIZED MALES receptor levels >10–15 fmol suggests that the cancer is probably steroid dependent and likely to respond to Androgen insensitivity actually presents itself as a antihormone therapy. However, breast cancer can spectrum of disorders ranging from complete external become estrogen independent and unresponsive to feminization to infertility in phenotypic males. A wide such as tamoxifen. A constitutively variety of AR gene defects have been documented rang- active ERα splice variant present in some breast ing from point mutations that cause a premature stop tumors may provide one explanation of how cancers codon in the testicular feminized male mouse to a com- can progress to a steroid-independent state. Another plete deletion of the AR gene in a human family. Known explanation may be that the overexpression of ER mutations within the human AR appear to cluster prima- coactivators, such as amplified in breast cancer-1, in rily within the DBD and SBD of the receptor. Generally, breast cancer cells results in increased levels of there is a reasonable correlation between the degree of coactivator activity that could reduce the effectiveness feminization and the degree to which normal function of of ER antagonists such as tamoxifen. the AR is altered, as assessed by various in vitro assays. For instance, mutations that totally abolish steroid bind- 6.2. Androgen-Based ing lead to profound feminization, and more subtle Developmental Defects mutations affecting thermolability and steroid dissocia- A number of different types of androgen-based defects tion rates lead to less profound effects such as infertility have been documented. These range from a defective and hypospadia. 5α-reductase enzyme that occurs as a rare autosomal Fertility problems related to AR defects are resis- mutation to a defective AR resulting from mutations tant to therapy whereas anomalies such as mild hypo- within the X-linked AR gene. In addition, alterations in spadia can be treated by surgical correction. In cases of steroidogenic enzymes earlier in the synthetic pathways complete feminization, inguinal and labial testes are can also result in developmental anomalies of andro- removed owing to increased incidences of testicular gen-dependent tissues. The phenotypic manifestations cancer. Infertile completely feminized XY individuals of these defects range from infertility in phenotypically develop female gender roles and tend to maintain these normal males to complete feminization of external geni- roles throughout adulthood. Chapter 4 / Steroid Hormones 63

6.3. Estrogen-Based Developmental Defects include underdeveloped external genitalia, uteri, and Until recently, no mutations in the aromatase or ER mammary glands, and infertility. α genes had been detected. Additionally, ER mRNA had 6.3.2. ESTROGEN RECEPTOR MUTATIONS been detected during very early embryonic stages using Recent work documented a normally masculinized, reverse transcriptase-polymerase chain reaction. Thus, human male with clinical symptoms very similar to it was suspected that estrogen is critical for develop- those of the aromatase-deficient male: tall stature, ment of a viable embryo and that mutations of either of incomplete epiphyseal closure, osteoporosis, decreased the aforementioned genes would be lethal. Recent find- sperm viability, and elevated testosterone and gonado- ings, however, have documented tropins. Estrogen levels were also elevated and the and estrogen insensitivity (ERα defects) in adult patient exhibited no response to E therapy. Molecular humans. In addition, gene-targeted mouse lines in 2 analysis revealed a point mutation that created a prema- which ERα(αERKO), ERβ (βERKO), or both ERα ture stop codon in the ERα gene resulting in a truncated and ERβ have been disrupted (αβERKO) demonstrate mutant form of the receptor protein. that embryos can develop in the absence of functional ERKO mice for both ERα and ERβ have been nuclear ER. Furthermore, aromatase-deficient mice developed to elucidate the role of ER-mediated signal- (ArKO), which lack the enzyme for converting andro- ing in normal growth and development. The female gens to estrogens, and therefore have no circulating αERKO mouse shows a number of interesting pheno- estrogens, are viable. Although these data suggest that types including reduced uterine development, absent estrogens may not be critical for embryonic survival, uterine responsiveness to E , mammary agenesis, hem- a number of phenotypic and receptor-specific abnor- 2 orrhagic cystic ovaries, anovulation, elevated gona- malities occur owing to these gene mutations. dotropins, and elevated testosterone and E2. Male 6.3.1. AROMATASE DEFICIENCY αERKO mice exhibit normal gross phenotype but are Mutations in the aromatase enzyme lead to alterations infertile as a result of reduced intromissions, reduced in phenotypes in both males and females. A male homo- sperm counts, and decreased sperm motility. The gross zygous for defective aromatase exhibited tall stature, sexual phenotype of the external genitalia and internal incomplete epiphyseal closure, continued linear bone androgen-dependent structures appears normal with growth, and osteoporosis. Circulating androgens and the exception of testicular dysmorphogenesis result- gonadotropin levels were increased but gross sexual ing in reduced testis size. In contrast to the complete phenotype was normal. In an aromatase-deficient female, infertility in αERKO mice, βERKO females are the individual presented with ambiguous genitalia at subfertile, have normal mammary gland development, birth but normal internal Müllerian structures by subse- and lactate normally, and males are fertile and have no quent laparoscopic examination. At puberty, the indi- testicular phenotype. vidual possessed the following symptoms: absence of (mammary agenesis), primary 6.4. Progesterone-Based amenorrhea, elevated gonadotropins, elevated andro- Developmental Defects gens, and polycystic ovaries. Estrogen treatment allevi- Defects in progesterone synthesis can arise owing to ated many of these symptoms. The masculinization is mutations in P450scc and 3β-HSD. However, because owing to a lack of conversion of C19 steroids into estro- of the pivotal position of progesterone in the synthetic gens and, hence, excess circulating androgens. pathways leading to other steroids, the consequences of As discussed previously, ArKO mice have been gen- the absence of progesterone synthesis are clouded by erated to characterize further the phenotypes associ- the absence of other important steroids. Thus, the con- ated with aromatase and estrogen deficiency. Many of sequences of defects in progesterone action may be more the phenotypes observed in ArKO mice are similar to accurately elucidated from cases involving a defective those seen in aromatase-deficient humans. Male ArKO PR. To this end, a PR knockout (PRKO) mouse that was mice exhibit an osteoporotic phenotype owing to a complete knockout of both isoforms of the PR (PR-A decreased bone formation, increased adipose tissue, and PR-B) was developed and characterized. The two elevated gonadotropin levels, age-dependent disrup- isoforms of the PR, PR-A (81 kDa) and PR-B (116 kDa), tions of spermatogenesis and fertility, and impaired are expressed from the same gene containing alternative sexual behavior. Female ArKO mice, like males, translation start sites. Female PRKO mice were anovu- exhibit an osteoporotic phenotype and increased latory, possessed underdeveloped mammary glands, and adiposity, as well as increased gonadotropin levels. did not display lordosis behavior. However, estrogen Reproductive phenotypes in the female ArKO mice treatments did cause uterine enlargement, hyperplasia, 64 Part II / Hormone Secretion and Action and , indicating a functional ER system. The male The most common form of adrenal hyperplasia is PRKO mouse was fertile and grossly normal except for owing to deficiency in 21-hydroxylase. The resulting an underdeveloped preputial gland. inability to synthesize cortisol leads to a buildup of pre- To elucidate the roles of the individual PR isoforms, cursors and subsequent conversion into testosterone. PR-A and PR-B knockout mice, designated PRAKO Clinically, females often present with ambiguous geni- and PRBKO, respectively, were developed and char- talia, irregular menstrual cycles, and some virilization. acterized. Phenotypic analyses have revealed that Both sexes undergo rapid somatic growth (postnatal), female PRAKO mice are infertile owing to severe accelerated skeletal growth, and early closure of epi- abnormalities in ovarian and uterine function. How- physeal plates. Some patients also have defects in the ever, PR-A-deficient mice have normal mammary ability to synthesize aldosterone, resulting in a “salt- gland and thymic responses to progesterone. By con- wasting” form of the disease. trast, PRBKO mice display normal ovarian, uterine, If the fetus is diagnosed prenatally via genotyping of and thymic responses but exhibit reduced mammary biopsy samples, the mother can undergo treatment with ductal morphogenesis. Taken together, these observa- to suppress excess production of andro- tions illustrate the distinct roles for each of the PR gens by the adrenal cortex of the fetus thus reducing isoforms in progesterone action. masculinization of female offspring. Neonatal screen- ing for 21-hydroxylase can be accomplished by assay- 6.5. Corticoid-Based ing for 17-hydroxyprogesterone. This may be useful in Developmental Defects preventing deaths related to salt-wasting forms of 21- Congenital adrenal hyperplasia is a heritable disor- hydroxylase deficiency. der in which the adrenal does not synthesize cortisol effectively. The inability to synthesize cortisol can 7. CONCLUSION result from defects in any of the enzymes involved in the Steroid hormones are synthesized and secreted by the synthesis of cortisol from cholesterol. As a consequence ovary, testis, adrenal, and placenta. The majority of ste- of these disorders, feedback inhibition of the pituitary is roidal effects occur through binding to specific intracel- absent and high levels of ACTH are secreted, resulting lular receptor proteins that regulate gene transcription in hypertrophy of the adrenal and, depending on the in target tissues. Steroids are critical for sexual differen- affected enzyme, high levels of precursors for cortisol tiation of different target organs and, in the adult, are synthesis. A large buildup of cortisol precursors can important regulators of many aspects of normal physi- result in synthesis of excess androgens and subsequent ology. Thus, heritable deficiencies in steroid synthesis masculinization of females. or receptor action often lead to permanent alterations in Deficiency in cholesterol desmolase (P450scc) differentiation of adult phenotype and, hence, altered leads to a deficiency in all steroid hormones and a function in the adult. Steroid-dependent tumors can syndrome referred to as congenital lipoid adrenal become steroid independent by somatic mutations in hyperplasia. Deficiency in 3β-hydroxysteroid dehy- receptor genes and alternate splicing of mRNAs. Recent drogenase also leads to a disorder in which synthesis of advances in molecular endocrinology and gene target- corticoids and sex steroids is deficient in adrenals and ing to generate relevant experimental animal models gonads. These disorders are characterized by an inabil- have allowed scientists to begin to elucidate the molecu- ity to produce cortisol and aldosterone and, hence, a lar mechanisms by which steroid hormones regulate reduced ability to regulate glucose metabolism, an normal physiology and pathophysiology. inability to conserve salt, and severe hyponatremia. In SELECTED READING males, the absence of sex steroids results in feminized external genitalia, and secretion of MIS by inguinal Clark JH, Mani SK. Actions of ovarian steroid hormones. In: Knobil E, Neil J, eds. The Physiology of Reproduction, vol. 1. New testes leads to regression of Mullerian derivatives. York, NY: Raven, 1988:1011–1059. Females with P450scc deficiencies are normal in Clark JH, Peck EJ, eds. Female Sex Steroids: Receptors and Func- appearance at birth but do not undergo pubertal tion. New York, NY: Springer-Verlag, 1979. β Couse JF, Curtis SW, Washburn TF, et al. Analysis of transcription changes. 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